Gene encoding Protein Cluster I and the encoded protein

ABSTRACT

The present invention relates to the identification of a human gene family expressed in metabolically relevant tissues. The genes encode a group polypeptides referred to as “Protein Cluster I” which are predicted to be useful in the diagnosis of metabolic diseases, such as obesity and diabetes, as well as in the identification of agents useful in the treatment of the said diseases.

TECHNICAL FIELD

[0001] The present invention relates to the identification of a human gene family expressed in metabolically relevant tissues. The genes encode a group polypeptides referred to as “Protein Cluster I” which are predicted to be useful in the diagnosis of metabolic diseases, such as obesity and diabetes, as well as in the identification of agents useful in the treatment of the said diseases.

BACKGROUND ART

[0002] Metabolic diseases are defined as any of the diseases or disorders that disrupt normal metabolism. They may arise from nutritional deficiencies; in connection with diseases of the endocrine system, the liver, or the kidneys; or as a result of genetic defects. Metabolic diseases are conditions caused by an abnormality in one or more of the chemical reactions essential to producing energy, to regenerating cellular constituents, or to eliminating unneeded products arising from these processes. Depending on which metabolic pathway is involved, a single defective chemical reaction may produce consequences that are narrow, involving a single body function, or broad, affecting many organs and systems.

[0003] One of the major hormones that influence metabolism is insulin, which is synthesized in the beta cells of the islets of Langerhans of the pancreas. Insulin primarily regulates the direction of metabolism, shifting many processes toward the storage of substrates and away from their degradation. Insulin acts to increase the transport of glucose and amino acids as well as key minerals such as potassium, magnesium, and phosphate from the blood into cells, It also regulates a variety of enzymatic reactions within the cells, all of which have a common overall direction, namely the synthesis of large molecules from small units. A deficiency in the action of insulin (diabetes mellitus) causes severe impairment in (i) the storage of glucose in the form of glycogen and the oxidation of glucose for energy; (ii) the synthesis and storage of fat from fatty acids and their precursors and the completion of fatty-acid oxidation; and (iii) the synthesis of proteins from amino acids.

[0004] There are two varieties of diabetes Type I is insulin-dependent diabetes mellitus (IDDM), for which insulin injection is required; it was formerly referred to as juvenile onset diabetes. In this type, insulin is not secreted by the pancreas and hence must be taken by injection. Type II, non-insulin-dependent diabetes mellitus (NIDDM) may be controlled by dietary restriction. It derives from insufficient pancreatic insulin secretion and tissue resistance to secreted insulin, which is complicated by subtle changes in the secretion of insulin by the beta cells. Despite their former classifications as juvenile or adult, either type can occur at any age; NIDDM, however, is the most common type, accounting for 90 percent of all diabetes. While the exact causes of diabetes remain obscure, it is evident that NIDDM is linked to heredity and obesity. There is clearly a genetic predisposition to NIDDM diabetes in those who become overweight or obese.

[0005] Obesity is usually defined in terms of the body mass index (BMI), i.e. weight (in kilograms) divided by the square of the height (in meters). Weight is regulated with great precision. Regulation of body weight is believed to occur not only in persons of normal weight but also among many obese persons, in whom obesity is attributed to an elevation in the set point around which weight is regulated. The determinants of obesity can be divided into genetic, environmental, and regulatory.

[0006] Recent discoveries have helped explain how genes may determine obesity and how they may influence the regulation of body weight. For example, mutations in the ob gene have led to massive obesity in mice. Cloning the ob gene led to the identification of leptin, a protein coded by this gene; leptin is produced in adipose tissue cells and acts to control body fat. The existence of leptin supports the idea that body weight is regulated, because leptin serves as a signal between adipose tissue and the areas of the brain that control energy metabolism, which influences body weight.

[0007] Metabolic diseases like diabetes and obesity are clinically and genetically heterogeneous disorders. Recent advances in molecular genetics have led to the recognition of genes involved in IDDM and in some subtypes of NIDDM, including maturity-onset diabetes of the young (MODY) (Velho & Froguel (1997) Diabetes Metab. 23 Suppl 2:34-37). However, several IDDM susceptibility genes have not yet been identified, and very little is known about genes contributing to common forms of NIDDM. Studies of candidate genes and of genes mapped in animal models of IDDM or NIDDM, as well as whole genome scanning of diabetic families from different populations, should allow the identification of most diabetes susceptibility genes and of the molecular targets for new potential drugs. The identification of genes involved in metabolic disorders will thus contribute to the development of novel predictive and therapeutic approaches.

BRIEF DESCRIPTION OF THE DRAWING

[0008]FIG. 1 Transmembrane regions identified in the proteins shown as (a) SEQ ID NO: 2; (fb) SEQ ID NO: 8; and (c) SEQ ID NO: 6.

DESCRIPTION OF THE INVENTION

[0009] According to the present invention, a family of genes and encoded homologous proteins (hereinafter referred to as “Protein Cluster I”) has been identified. Consequently, the present invention provides an isolated nucleic acid molecule selected from:

[0010] (a) nucleic acid molecules comprising a nucleotide sequence as shown in SEQ ID NO: 1,3,5 or7;

[0011] (b) nucleic acid molecules comprising a nucleotide sequence capable of hybridizing, under stringent hybridization conditions, to a nucleotide sequence complementary to the polypeptide coding region of a nucleic acid molecule as defined in (a); and

[0012] (c) nucleic acid molecules comprising a nucleic acid sequence which is degenerate as a result of the genetic code to a nucleotide sequence as defined in (a) or (b).

[0013] The nucleic acid molecules according to the present invention includes cDNA, chemically synthesized DNA, DNA isolated by PCR, genomic DNA, and combinations thereof. RNA transcribed from DNA is also encompassed by the present invention.

[0014] The term “stringent hybridization conditions” is known in the art from standard protocols (e.g. Ausubel et al., supra) and could be understood as e.g. hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at +65° C., and washing in 0.1×SSC/0.1% SDS at +68° C.

[0015] In a preferred form of the invention, the said nucleic acid molecule has a nucleotide sequence identical with SEQ ID NO: 1 of the Sequence Listing. However, the nucleic acid molecule according to the invention is not to be limited strictly to the sequence shown as SEQ ID NO: 1. Rather the invention encompasses nucleic acid molecules carrying modifications like substitutions, small deletions, insertions or inversions, which nevertheless encode proteins having substantially the features of the Protein Cluster I polypeptide according to the invention. Included in the invention are consequently nucleic acid molecules, the nucleotide sequence of which is at least 90% homologous, preferably at least 95% homologous, with the nucleotide sequence shown as SEQ ID NO: 1 in the Sequence Listing.

[0016] Included in the invention is also a nucleic acid molecule which nucleotide sequence is degenerate, because of the genetic code, to the nucleotide sequence shown as SEQ ID NO: 1. A sequential grouping of three nucleotides, a “codon”, codes for one amino acid. Since there are 64 possible codons, but only 20 natural amino acids, most amino acids are coded for by more than one codon. This natural “degeneracy”, or “redundancy”, of the genetic code is well known in the art. It will thus be appreciated that the nucleotide sequence shown in the Sequence Listing is only an example within a large but definite group of sequences which will encode the Protein Cluster I polypeptide.

[0017] The nucleic acid molecules according to the invention have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include their use as hybridization probes, for chromosome and gene mapping, in PCR technologies, in the production of sense or antisense nucleic acids, in screening for new therapeutic molecules, etc.

[0018] More specifically, the sequence information provided by the invention makes possible large-scale expression of the encoded polypeptides by techniques well known in the art. Nucleic acid molecules of the invention also permit identification and isolation of nucleic acid molecules encoding related polypeptides, such as human allelic variants and species homologues, by well-known techniques including Southern and/or Northern hybridization, and PCR. Knowledge of the sequence of a human DNA also makes possible, through use of Southern hybridization or PCR, the identification of genomic DNA sequences encoding the proteins in Cluster I, expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like. Nucleic acid molecules of the invention are also useful in hybridization assays to detect the capacity of cells to express the proteins in Cluster I. Nucleic acid molecules of the invention may also provide a basis for diagnostic methods useful for identifying a genetic alteration(s) in a locus that underlies a disease state or states, which information is useful both for diagnosis and for selection of therapeutic strategies.

[0019] In a further aspect, the invention provides an isolated polypeptide encoded by the nucleic acid molecule as defined above. In a preferred form, the said polypeptide has an amino acid sequence according to SEQ ID NO: 2, 4, 6 or 8 of the Sequence Listing. However, the polypeptide according to the invention is not to be limited strictly to a polypeptide with an amino acid sequence identical with SEQ ID NO: 2, 4, 6 or 8 in the Sequence Listing. Rather the invention encompasses polypeptides carrying modifications like substitutions, small deletions, insertions or inversions, which polypeptides nevertheless have substantially the features of the Protein Cluster I polypeptide. Included in the invention are consequently polypeptides, the amino acid sequence of which is at least 90% homologous, preferably at least 95% homologous, with the amino acid sequence shown as SEQ ID NO: 2, 4, 6 or 8 in the Sequence Listing.

[0020] In a further aspect, the invention provides a vector harboring the nucleic acid molecule as defined above. The said vector can e.g. be a replicable expression vector, which carries and is capable of mediating the expression of a DNA molecule according to the invention. In the present context the term “replicable” means that the vector is able to replicate in a given type of host cell into which is has been introduced. Examples of vectors are viruses such as bacteriophages, cosmids, plasmids and other recombination vectors. Nucleic acid molecules are inserted into vector genomes by methods well known in the art.

[0021] Included in the invention is also a cultured host cell harboring a vector according to the invention. Such a host cell can be a prokaryotic cell, a unicellular eukaryotic cell or a cell derived from a multicellular organism. The host cell can thus e.g. be a bacterial cell such as an E. coli cell; a cell from a yeast such as Saccharomyces cervisiae or Pichia pastoris, or a mammalian cell. The methods employed to effect introduction of the vector into the host cell are standard methods well known to a person familiar with recombinant DNA methods.

[0022] In yet another aspect, the invention provides a process for production of a polypeptide, comprising culturing a host cell, according to the invention, under conditions whereby said polypeptide is produced, and recovering said polypeptide. The medium used to grow the cells may be any conventional medium suitable for the purpose. A suitable vector may be any of the vectors described above, and an appropriate host cell may be any of the cell types listed above. The methods employed to construct the vector and effect introduction thereof into the host cell may be any methods known for such purposes within the field of recombinant DNA. The recombinant polypeptide expressed by the cells may be secreted, i.e. exported through the cell membrane, dependent on the type of cell and the composition of the vector.

[0023] In a further aspect, the invention provides a method for identifying an agent capable of modulating a nucleic acid molecule according to the invention, comprising

[0024] (i) providing a cell comprising the said nucleic acid molecule;

[0025] (ii) contacting said cell with a candidate agent; and

[0026] (iii) monitoring said cell for an effect that is not present in the absence of said candidate agent.

[0027] For screening purposes, appropriate host cells can be transformed with a vector having a reporter gene under the control of the nucleic acid molecule according to this invention. The expression of the reporter gene can be measured in the presence or absence of an agent with known activity (i.e. a standard agent) or putative activity (i.e. a “test agent” or “candidate agent”). A change in the level of expression of the reporter gene in the presence of the test agent is compared with that effected by the standard agent. In this way, active agents are identified and their relative potency in this assay determined.

[0028] A transfection assay can be a particularly useful screening assay for identifying an effective agent. In a transfection assay, a nucleic acid containing a gene such as a reporter gene that is operably linked to a nucleic acid molecule according to the invention, is transfected into the desired cell type. A test level of reporter gene expression is assayed in the presence of a candidate agent and compared to a control level of expression. An effective agent is identified as an agent that results in a test level of expression that is different than a control level of reporter gene expression, which is the level of expression determined in the absence of the agent. Methods for transfecting cells and a variety of convenient reporter genes are well known in the art (see, for example, Goeddel (ed.), Methods Enzymol., Vol. 185, San Diego: Academic Press, Inc. (1990); see also Sambrook, supra).

[0029] Throughout this description the terms “standard protocols” and “standard procedures”, when used in the context of molecular biology techniques, are to be understood as protocols and procedures found in an ordinary laboratory manual such as: Current Protocols in Molecular Biology, editors F. Ausubel et al., John Wiley and Sons, Inc. 1994, or Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning. A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989.

[0030] Additional features of the invention will be apparent from the following Examples. Examples 1 to 3 are actual, while Examples 4 to 9 are prophetic.

EXAMPLES Example 1

[0031] Identification of protein clusters

[0032] A family of homologous proteins (hereinafter referred to as “Protein Cluster I”) was identified by an “all-versus-all” BLAST procedure using all Caenorhabditis elegans proteins in the Wormpep2o database release (http://www.sanger. ac. uk/Projects/C_elegans/wormpep/index.shtm/). The Wormpep database contains the predicted proteins from the C. elegans genome sequencing project, carried out jointly by the Sanger Centre in Cambridge, UK and the Genome Sequencing Center in St. Louis, USA. A number of 18,940 proteins were retrieved from Wormpep20. The proteins were used in a Smith-Waterman clustering procedure to group together proteins of similarity (Smith T. F. & Waterman M. S. (1981) Identification of common molecular subsequences. J. Mol. Biol. 147(1): 195-197; Pearson W R. (1991) Searching protein sequence libraries: comparison of the sensitivity and selectivity of the Smith-Waterman andFASTA algorithms. Genomics 11: 635-650; Olsen et al. (1999) Optimizing Smith-Waterman alignments. Pac Symp Biocomput.302-313). Completely annotated proteins were filtered out, whereby 10,130 proteins of unknown function could be grouped into 1,800 clusters.

[0033] The obtained sequence clusters were compared to the Drosophila melanogaster proteins contained in the database Flybase (Berkeley Drosophila Genome Project; http://www.fruitfly. org), and annotated clusters were removed. Non-annotated protein clusters, conserved in both C. elegans and D. melanogaster, were saved to a worm/fly data set, which was used in a BLAST procedure (http://www. ncbi. nlm. nih. gov/Education/BLASTinfo/information3.htmr) against the Celera Human Genome Database (http:l/www. celera. com). Overlapping fragments were assembled to, as close as possible, full-length proteins using the PHRAP software, developed at the University of Washington (http.//www. genome. Washington. edu/UWGC/analysistools/phrap. htm). A group of homologous proteins (“Protein Cluster I”) with unknown function was chosen for further studies.

Example 2

[0034] Analyses of Protein Cluster I

[0035] (a) Alignment

[0036] The human part of Protein Cluster I comprises polypeptides encoded by three genes (SEQ ID NOS: 19 5 and 7). In addition, an alternative splicing (corresponding to a deletion of positions 624 to 794 of the gene shown as SEQ ID NO: 1 results in SEQ ID NO: 3. The gene shown as SEQ ID NO: 1 was found to be comprised in a human DNA sequence from clone RP11-108L7 on chromosome 10 (GenBank Accession No. AL133215).

[0037] An alignment of the human polypeptides included in Protein Cluster I (SEQ ID NOS: 2, 4, 6 and 8), using the ClustalX multiple alignment software (downloadable from e.g. ftp:llftp. ebi. ac. uk) is shown in Table I. For references to the ClustalX software, see Thompson et al. (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools; Nucleic Acids Research, 24:4876-4882. See also Jeanmougin et al. (1998) Multiple sequence alignment with ClustalX; Trends Biochem. Sci. 23:403-405. The alignment showed a high degree of conservation in two separate regions, indicating the presence of two novel domains (see positions marked with stars in Table I).

[0038] (b) HMM-Pfam

[0039] A HMM-Pfam search was performed on the three human family members. Pfam (http://pfam. wustl. edu) is a large collection of protein families and domains. Pfam contains multiple protein alignments and profile-HMMs (Profile Hidden Markov Models) of these families. Profile-HMMs can be used to do sensitive database searching using statistical descriptions of a sequence family's consensus. Pfam is available on the WWW at http://pfam.wustl.edu; http.//www.sanger.ac.ukISoftware/Pfam; and http://www.cgr.ki.se/Pfam. The latest version (4.3) of Pfam contains 1815 families. These Pfam families match 63% of proteins in SWISS-PROT 37 and TrEMBL 9. For references to Pfam, see Bateman et al. (2000) The Pfam protein families database. Nucleic Acids Res. 28:263-266; Sonnhammer et al. (1998) Pfam: Multiple Sequence Alignments and HMM-Profiles ofProtein Domains. Nucleic Acids Research, 26:322-325; Sonnhammer et al. (1997) Pfam: a Comprehensive Database off Protein Domain Families Based on Seed Alignments. Proteins 28:405-420.

[0040] The HMM-Pfam search indicated that no previously known domains could be identified in Protein Cluster I.

[0041] (c) TM-HMM

[0042] The human proteins in Cluster I were analyzed using the TM-HMM tool available at http://www.cbs.dtu.dklservices/TMHMM-1.O. TM-HMM is a method to model and predict the location and orientation of alpha helices in membrane-spanning proteins (Sonnhammer et al. (1998) A hidden Markov model for predicting transmembrane helices in protein sequences. ISMB 6:175-182). Transmembrane segments were identified in the proteins shown as SEQ ID NOS: 2, 6 and 8 (FIG. 1)

[0043] (d) Analysis ofnon-human orthologs

[0044] The C. elegans genome includes six genes encoding proteins within Protein Cluster I, of which the closest ancestor in evolution, a sequence included the C. elegans cosmid T04F8.1 (GenBank Accession No. Z66565; see also: Genome sequence of the nematode C. elegans: a platform for investigating biology; The C. elegans Sequencing Consortium. Science (1998) 282:2012-2018. Published errata appear in Science (1999) 283:35; 283:2103; and 285:1493.) is 53% identical to the three identified human proteins (SEQ ID NOS: 2, 6 and 8).

[0045] The Drosophila melanogaster genome comprises two genes belonging to Protein Cluster I, of which the closest relative (GenBank Accession No. AE003606 24; see also Adams et al. (2000) The genome sequence ofDrosophila melanogaster; Science 287:2185-2195) is 53% identical to the human protein set.

[0046] The human proteins also show 38% identity to a Saccharomyces cerevisiae protein (GenPept Accession No. CAA99495. 1). The yeast protein has been annotated in the Saccharomyces Genome Database as a putative transporter (http://genome-www4.stanford edu/cgi-bin/SGD/locus.pl?locus=YOR2 70c).

[0047] Two public Rattus norvegicus database entries (GENBANK entries AF276997 and S70011) have been annotated as putative tricarboxylate carrier proteins. The genes have 88% and 79% identity, respectively, with SEQ ID NO: 1. The tricarboxylate carrier transports citrate or other tricarboxylates across the inner membranes of mitochondria in an electroneutral exchange for malate or other dicarboxylic acids. (Azzi et al. (1993) J. Bioenerg. Biomembr. 25: 515-524).

Example 3

[0048] Expression analysis

[0049] EST databases provided by the EMBL (http://www. embl. org/Services/index. html) were used to check whether the human proteins in Cluster I were expressed, in order to identify putative pseudogenes. No putative pseudogenes were identified in Protein Cluster I.

[0050] The tissue distribution of the human genes was studied using the Incyte LifeSeq® database (http://www. incyte. com). The nucleic acid molecule shown as SEQ ID NO: 1 was found to be expressed primarily in the nervous system and the digestive system. The nucleic acid molecule shown as SEQ ID NO: 3 was expressed primarily in male genitalia. The nucleic acid molecule shown as SEQ ID NO: 5 was expressed primarily in the liver and in embryonic structures. The nucleic acid molecule shown as SEQ ID NO: 7 was expressed primarily in the immune system. Therefore, the said nucleic acid molecules shown as SEQ ID NO: 1, 3, 5 and 7 and the polypeptides shown as SEQ ID NO: 2, 4, 6 and 8 are proposed to be useful for differential identification of the tissue(s) or cell types(s) present in a biological sample and for diagnosis of diseases and disorders, including metabolic disorders and immune disorders.

Example 4

[0051] Multiple Tissue Northern blotting

[0052] Multiple Tissue Northern blotting (MTN) is performed to make a more thorough analysis of the expression profiles of the proteins in Cluster I. Multiple Tissue Northern (MTNTM) Blots (http://www. clontech. com/mtn) are pre-made Northern blots featuring Premium Poly A+RNA from a variety of different human, mouse, or rat tissues. MTN Blots can be used to analyze size and relative abundance of transcripts in different tissues. MTN Blots can also be used to investigate gene families and alternate splice forms and to assess cross species homology.

Example 5

[0053] Expressing profiling using microarrays

[0054] Microarrays consist of a highly ordered matrix of thousands of different DNA sequences that can be used to measure DNA and RNA variation in applications that include gene expression profiling, comparative genomics and genotyping (For recent reviews, see e.g.: Harrington et al. (2000) Monitoring gene expression using DNA microarrays. Curr. Opin. Microbiol. 3(3): 285-291; or Duggan et al. (1999) Expression profiling using cDNA Microarrays. Nature Genetics Supplement 21:10-14).

[0055] The expression pattern of the proteins in Cluster I can be analyzed using GeneChip® expression arrays (http.//www.affymetrix.com/products/app_exp.html). Briefly, mRNAs are extracted from various tissues. They are reverse transcribed using a T7-tagged oligo-dT primer and double-stranded cDNAs are generated. These cDNAs are then amplified and labeled using In Vitro Transcription (IVT) with T7 RNA polymerase and biotinylated nucleotides. The populations of cRNAs obtained are purified and fragmented by heat to produce a distribution of RNA fragment sizes from approximately 35 to 200 bases. GeneChip® expression arrays are hybridized with the samples. The arrays are washed and stained. The cartridges are scanned using a confocal scanner and the images are analyzed with the GeneChip 3.1 software (Affymetrix).

Example 6

[0056] Identification of polypeptides binding to Protein Cluster I

[0057] In order to assay for proteins interacting with Protein Cluster I, the two-hybrid screening method can be used. The two-hybrid method, first described by Fields & Song (1989) Nature 340:245-247, is a yeast-based genetic assay to detect protein-protein interactions in vivo. The method enables not only identification of interacting proteins, but also results in the immediate availability of the cloned genes for these proteins.

[0058] The two-hybrid method can be used to determine if two known proteins (i.e. proteins for which the corresponding genes have been previously cloned) interact. Another important application of the two-hybrid method is to identify previously unknown proteins that interact with a target protein by screening a two-hybrid library. For reviews, see e.g.: Chien et al. (1991) The two-hybrid system, a method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. U.S.A. 88:9578-9582; Bartel P L, Fields (1995) Analyzing protein-protein interactions using two-hybrid system. Methods Enzymol. 254:241-263; or Wallach et al. (1998) The yeast two-hybrid screening technique and its use in the study of protein-protein interactions in apoptosis. Curr. Opin. Immunol. 10(2): 131-136. See also http://www. clontech. com/matchmaker.

[0059] The two-hybrid method uses the restoration of transcriptional activation to indicate the interaction between two proteins. Central to this technique is the fact that many eukaryotic transcriptional activators consist of two physically discrete modular domains: the DNA-binding domain (DNA-BD) that binds to a specific promoter sequence and the activation domain (AD) that directs the RNA polymerase II complex to transcribe the gene downstream of the DNA binding site. The DNA-BD vector is used to generate a fusion of the DNA-BD and a bait protein X, and the AD vector is used to generate a fusion of the AD and another protein Y. An entire library of hybrids with the AD can also be constructed to search for new or unknown proteins that interact with the bait protein. When interaction occurs between the bait protein X and a candidate protein Y, the two functional domains, responsible for DNA binding and activation, are tethered, resulting in functional restoration of transcriptional activation. The two hybrids are cotransformed into a yeast host strain harboring reporter genes containing appropriate upstream binding sites; expression of the reporter genes then indicates interaction between a candidate protein and the target protein.

Example 7

[0060] Full-length cloning of Cluster I genes

[0061] The polymerase chain reaction (PCR), which is a well known procedure for in vitro enzymatic amplification of a specific DNA segment, can be used for direct cloning of Protein Cluster I genes. Tissue cDNA can be amplified by PCR and cloned into an appropriate plasmid and sequenced. For reviews, see e.g. Hooft van Huijsduijnen (1998) PCR-assisted cDNA cloning: a guided tour of the minefield. Biotechniques 24:390-392; Lenstra (1995) The applications of the polymerase chain reaction in the life sciences. Cellular & Molecular Biology 41:603-614; or Rashtchian (1995) Novel methods for cloning and engineering genes using the polymerase chain reaction. Current Opinion in Biotechnology 6:30-36. Various methods for generating suitable ends to facilitate the direct cloning of PCR products are given e.g. in Ausubel et al. supra (section 15.7).

[0062] In an alternative approach to isolate a cDNA clone encoding a full length protein of Protein Cluster I, a DNA fragment corresponding to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 5 or 7, or a portion thereof, can be used as a probe for hybridization screening of a phage cDNA library. The DNA fragment is arnplified by the polymerase chain reaction (PCR) method. The primers are preferably 10 to 25 nucleotides in length and are determined by procedures well known to those skilled in the art. A lambda phage library containing cDNAs cloned into lambda phage-vectors is plated on agar plates with E. coli host cells, and grown. Phage plaques are transferred to nylon membranes, which are hybridized with a DNA probe prepared as described above. Positive colonies are isolated from the plates. Plasmids containing cDNA are rescued from the isolated phages by standard methods. Plasmid DNA is isolated from the clones. The size of the insert is determined by digesting the plasmid with appropriate restriction enzymes. The sequence of the entire insert is determined by automated sequencing of the plasmids.

Example 8

[0063] Recombinant expression of proteins in eukaryotic host cells

[0064] To produce proteins of Cluster I, a polypeptide-encoding nucleic acid molecule is expressed in a suitable host cell using a suitable expression vector and standard genetic engineering techniques. For example, the polypeptide-encoding sequence is subdloned into a commercial expression vector and transfected into mammalian, e.g. Chinese Hamster Ovary (CHO), cells using a standard transfection reagent. Cells stably expressing a protein are selected. Optionally, the protein may be purified from the cells using standard chromatographic techniques. To facilitate purification, antisera is raised against one or more synthetic peptide sequences that correspond to portions of the amino acid sequence, and the antisera is used to affinity purify the protein.

Example 9

[0065] Determination of gene function

[0066] Methods are known in the art for elucidating the biological function or mode of action of individual genes. For instance, RNA interference (RNAi) offers a way of specifically and potently inactivating a cloned gene, and is proving a powerful tool for investigating zzr. gene finction. For reviews, see e.g. Fire (1999) RNA-triggered gene silencing. Trends in Genetics 15:358-363; or Kuwabara & Coulson (2000) RNATi-prospects for a general techniquefor determining gene function. Parasitology Today 16:347-349. When double-stranded RNA (dsRNA) corresponding to a sense and antisense sequence of an endogenous mRNA is introduced into a cell, the cognate mRNA is degraded and the gene is silenced. This type of post transcriptional gene silencing (PTGS) was first discovered in C. elegans (Fire et al., (1998) Nature 391:806-811). RNA interference has recently been used for targeting nearly 90% of predicted genes on C. elegans chromosome I (Fraser et al. (2000) Nature 408: 325-330) and 96% of predicted genes on C. elegans chromosome III (Gönczy et al. (2000) Nature 408:331-336). TABLE I Alignment of polypeptides in Protein Cluster I        *    **  ***** *****  **  ***** * **  **   * ** SEQ ID NO: 2 MESKMGELPLDINIQEPRWDQSTFLGRARHFFTVTDPRNLLLSGAQLEASRNIVQNYRAGVVTPGITEDQLWRAKY (76) SEQ ID NO: 4 MESKMGELPLDINIQEPRWDQSTFLGRARHFFTVTDPRNLLLSGAQLEASRNIVQNY------------------- (57) SEQ ID NO: 6 ---MEADLSG-FNIDAPRWDQRTFLGRVKHFLNITDPRTVFVSERELDWAKVMVEKSRMGVVPPGTQVEQLLYAKK (72) SEQ ID NO: 8 ---MSGELPPNINIKEPRWDQSTFIGRANHFFTVTDPRNILLTNEQLESARKIVHDYRQGIVPPGLTENELWRAKY (73)                                       *  * *************** SEQ ID NO: 2 VYDSAFHPDTGEKVVLIGRMSAQVPMNMTITGCMLTFYRKTPTVVFWQWVNQSFNAIVNYSNRSGDTPITVRQLGT (152) SEQ ID NO: 4 --------------------------------------RKTPTVVFWQWVNQSFNAIV------------------ (77) SEQ ID NO: 6 LYDSAFHPDTGEKMNVIGRMSFQLPGGMIITGFMLQFYRTMPAVIFWQWVNQSFNALVNYTNRNAASPTSVRQMAL (148) SEQ ID NO: 8 IYDSAFHPDTGEKMILIGRMSAQVPMNMTITGCMMTFYRTTPAVLFWQWINQSFNAVVNYTNRSGDAPLTVNELGT (149) SEQ ID NO: 2 AYVSATTGAVATALGLKSLTKHLPPLVGRFVPFAAVAAANCINIPLNRQRELQVGIPVADEAGQRLGYSVTAAKQG (228) SEQ ID NO: 4 ---------------------------------------------------------------------------- SEQ ID NO: 6 SYFTATTTAVATAVGMNMLTKKAPPLVGRWVPFAAVAAANCVNIPMNRQRELIKGICVKDRNENEIGHSRRAAAIG (224) SEQ ID NO: 8 AYVSATTGAVATALGLNALTKHVSPLIGRFVPFAAVAAANCINIPLNRQRELKVGIPVTDENGNRLGESANAAKQA (225) SEQ ID NO: 2 IFQVVISRICMAIPAMAIPPLIMDTLEKKDFLK-------------------------------------------------- (261) --------------- SEQ ID NO: 4 ----------------------------------------------------------------------------------- --------------- SEQ ID NO: 6 ITQVVISRITMSAPGMILLPVIMERLEKLHFMQKVKVLHAPLQVMLSGCFLIFMVPVACGLFPQKCELPVSYLEPKLQDTIKA (300) KYGELEPYVYFNKGL SEQ ID NO: 8 ITQVVVSRILMAAPGMAIPPFIMNTLEKKAFLKRFPWMSAPIQVGLVGFCLVFATPLCCALFPQKSSMSVTSLEAELQAKIQE (301) SHPELR-RVYFNKGL

[0067]

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 8 <210> SEQ ID NO 1 <211> LENGTH: 1232 <212> TYPE: DNA <213> ORGANISM: human <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (450)..(1232) <400> SEQUENCE: 1 cccttaggcg ccagggacag ccgagcgtta cctggtcccg ggcagcggag ttctttaccc 60 accccagttc tggttctgac gccctagctc attccgcaaa tttagggctt gggtctggct 120 tgttcccctc cggctcgaac cacctcttct ctgagccgag ccagctaccg gggctcctgg 180 aattgccacc cctccctggg cacccttgag gcctccgtgg agggacgtca cggggcagag 240 cgggacgtga gcctgagttt gctgcaggcg tgctctgtgt ggtggctggg ttctgccaat 300 ccccgtgccc accgggtggg cgcggccggg aagctcctgc ccctccctgc tggtcggcgt 360 cacgcgtgac gtcccgcgtg atggctggga gggcccggcg gcgacagcgg aggcagagag 420 gaaggcggtt ctgagagctt cagagagcg atg gaa agc aaa atg ggt gaa ttg 473 Met Glu Ser Lys Met Gly Glu Leu 1 5 cct tta gac atc aac atc cag gaa cct cgc tgg gac caa agt act ttc 521 Pro Leu Asp Ile Asn Ile Gln Glu Pro Arg Trp Asp Gln Ser Thr Phe 10 15 20 ctg ggc aga gcc cgg cac ttt ttc act gtt act gat cct cga aat ctg 569 Leu Gly Arg Ala Arg His Phe Phe Thr Val Thr Asp Pro Arg Asn Leu 25 30 35 40 ctg ctg tcc ggg gca cag ctg gaa gct tct cgg aac atc gtg cag aac 617 Leu Leu Ser Gly Ala Gln Leu Glu Ala Ser Arg Asn Ile Val Gln Asn 45 50 55 tac agg gcc ggc gtg gtg acc cca ggg atc acc gag gac cag ctg tgg 665 Tyr Arg Ala Gly Val Val Thr Pro Gly Ile Thr Glu Asp Gln Leu Trp 60 65 70 agg gcc aag tat gtg tat gac tcc gcc ttc cat ccg gac aca ggg gag 713 Arg Ala Lys Tyr Val Tyr Asp Ser Ala Phe His Pro Asp Thr Gly Glu 75 80 85 aag gtg gtc ctg att ggc cgc atg tca gcc cag gtg ccc atg aac atg 761 Lys Val Val Leu Ile Gly Arg Met Ser Ala Gln Val Pro Met Asn Met 90 95 100 acc atc act ggc tgc atg ctc aca ttc tac agg aag acc cca acc gtg 809 Thr Ile Thr Gly Cys Met Leu Thr Phe Tyr Arg Lys Thr Pro Thr Val 105 110 115 120 gtg ttc tgg cag tgg gtg aat cag tcc ttc aat gcc att gtt aac tac 857 Val Phe Trp Gln Trp Val Asn Gln Ser Phe Asn Ala Ile Val Asn Tyr 125 130 135 tcc aac cgc agt ggt gac act ccc atc act gtg agg cag ctg ggg aca 905 Ser Asn Arg Ser Gly Asp Thr Pro Ile Thr Val Arg Gln Leu Gly Thr 140 145 150 gcc tat gtg agt gcc acc act gga gct gtg gcc acg gcc ctg gga ctc 953 Ala Tyr Val Ser Ala Thr Thr Gly Ala Val Ala Thr Ala Leu Gly Leu 155 160 165 aaa tcc ctc acc aag cac ctg ccc ccc ttg gtc ggc aga ttt gtg ccc 1001 Lys Ser Leu Thr Lys His Leu Pro Pro Leu Val Gly Arg Phe Val Pro 170 175 180 ttt gca gca gtg gca gct gcc aac tgc atc aac atc ccc ctg atg agg 1049 Phe Ala Ala Val Ala Ala Ala Asn Cys Ile Asn Ile Pro Leu Met Arg 185 190 195 200 cag aga gag ctg cag gtg ggc atc ccg gtg gct gat gag gca ggt cag 1097 Gln Arg Glu Leu Gln Val Gly Ile Pro Val Ala Asp Glu Ala Gly Gln 205 210 215 agg ctt ggc tac tcg gtg act gca gcc aag cag gga atc ttc cag gtg 1145 Arg Leu Gly Tyr Ser Val Thr Ala Ala Lys Gln Gly Ile Phe Gln Val 220 225 230 gtg att tca aga atc tgc atg gcg att cct gcc atg gcc atc cca cca 1193 Val Ile Ser Arg Ile Cys Met Ala Ile Pro Ala Met Ala Ile Pro Pro 235 240 245 ctg atc atg gac act ctg gag aag aaa gac ttc ctg aag 1232 Leu Ile Met Asp Thr Leu Glu Lys Lys Asp Phe Leu Lys 250 255 260 <210> SEQ ID NO 2 <211> LENGTH: 261 <212> TYPE: PRT <213> ORGANISM: human <400> SEQUENCE: 2 Met Glu Ser Lys Met Gly Glu Leu Pro Leu Asp Ile Asn Ile Gln Glu 1 5 10 15 Pro Arg Trp Asp Gln Ser Thr Phe Leu Gly Arg Ala Arg His Phe Phe 20 25 30 Thr Val Thr Asp Pro Arg Asn Leu Leu Leu Ser Gly Ala Gln Leu Glu 35 40 45 Ala Ser Arg Asn Ile Val Gln Asn Tyr Arg Ala Gly Val Val Thr Pro 50 55 60 Gly Ile Thr Glu Asp Gln Leu Trp Arg Ala Lys Tyr Val Tyr Asp Ser 65 70 75 80 Ala Phe His Pro Asp Thr Gly Glu Lys Val Val Leu Ile Gly Arg Met 85 90 95 Ser Ala Gln Val Pro Met Asn Met Thr Ile Thr Gly Cys Met Leu Thr 100 105 110 Phe Tyr Arg Lys Thr Pro Thr Val Val Phe Trp Gln Trp Val Asn Gln 115 120 125 Ser Phe Asn Ala Ile Val Asn Tyr Ser Asn Arg Ser Gly Asp Thr Pro 130 135 140 Ile Thr Val Arg Gln Leu Gly Thr Ala Tyr Val Ser Ala Thr Thr Gly 145 150 155 160 Ala Val Ala Thr Ala Leu Gly Leu Lys Ser Leu Thr Lys His Leu Pro 165 170 175 Pro Leu Val Gly Arg Phe Val Pro Phe Ala Ala Val Ala Ala Ala Asn 180 185 190 Cys Ile Asn Ile Pro Leu Met Arg Gln Arg Glu Leu Gln Val Gly Ile 195 200 205 Pro Val Ala Asp Glu Ala Gly Gln Arg Leu Gly Tyr Ser Val Thr Ala 210 215 220 Ala Lys Gln Gly Ile Phe Gln Val Val Ile Ser Arg Ile Cys Met Ala 225 230 235 240 Ile Pro Ala Met Ala Ile Pro Pro Leu Ile Met Asp Thr Leu Glu Lys 245 250 255 Lys Asp Phe Leu Lys 260 <210> SEQ ID NO 3 <211> LENGTH: 1061 <212> TYPE: DNA <213> ORGANISM: human <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (450)..(680) <400> SEQUENCE: 3 cccttaggcg ccagggacag ccgagcgtta cctggtcccg ggcagcggag ttctttaccc 60 accccagttc tggttctgac gccctagctc attccgcaaa tttagggctt gggtctggct 120 tgttcccctc cggctcgaac cacctcttct ctgagccgag ccagctaccg gggctcctgg 180 aattgccacc cctccctggg cacccttgag gcctccgtgg agggacgtca cggggcagag 240 cgggacgtga gcctgagttt gctgcaggcg tgctctgtgt ggtggctggg ttctgccaat 300 ccccgtgccc accgggtggg cgcggccggg aagctcctgc ccctccctgc tggtcggcgt 360 cacgcgtgac gtcccgcgtg atggctggga gggcccggcg gcgacagcgg aggcagagag 420 gaaggcggtt ctgagagctt cagagagcg atg gaa agc aaa atg ggt gaa ttg 473 Met Glu Ser Lys Met Gly Glu Leu 1 5 cct tta gac atc aac atc cag gaa cct cgc tgg gac caa agt act ttc 521 Pro Leu Asp Ile Asn Ile Gln Glu Pro Arg Trp Asp Gln Ser Thr Phe 10 15 20 ctg ggc aga gcc cgg cac ttt ttc act gtt act gat cct cga aat ctg 569 Leu Gly Arg Ala Arg His Phe Phe Thr Val Thr Asp Pro Arg Asn Leu 25 30 35 40 ctg ctg tcc ggg gca cag ctg gaa gct tct cgg aac atc gtg cag aac 617 Leu Leu Ser Gly Ala Gln Leu Glu Ala Ser Arg Asn Ile Val Gln Asn 45 50 55 tac agg aag acc cca acc gtg gtg ttc tgg cag tgg gtg aat cag tcc 665 Tyr Arg Lys Thr Pro Thr Val Val Phe Trp Gln Trp Val Asn Gln Ser 60 65 70 ttc aat gcc att gtt aactactcca accgcagtgg tgacactccc atcactgtga 720 Phe Asn Ala Ile Val 75 ggcagctggg gacagcctat gtgagtgcca ccactggagc tgtggccacg gccctgggac 780 tcaaatccct caccaagcac ctgcccccct tggtcggcag atttgtgccc tttgcagcag 840 tggcagctgc caactgcatc aacatccccc tgatgaggca gagagagctg caggtgggca 900 tcccggtggc tgatgaggca ggtcagaggc ttggctactc ggtgactgca gccaagcagg 960 gaatcttcca ggtggtgatt tcaagaatct gcatggcgat tcctgccatg gccatcccac 1020 cactgatcat ggacactctg gagaagaaag acttcctgaa g 1061 <210> SEQ ID NO 4 <211> LENGTH: 77 <212> TYPE: PRT <213> ORGANISM: human <400> SEQUENCE: 4 Met Glu Ser Lys Met Gly Glu Leu Pro Leu Asp Ile Asn Ile Gln Glu 1 5 10 15 Pro Arg Trp Asp Gln Ser Thr Phe Leu Gly Arg Ala Arg His Phe Phe 20 25 30 Thr Val Thr Asp Pro Arg Asn Leu Leu Leu Ser Gly Ala Gln Leu Glu 35 40 45 Ala Ser Arg Asn Ile Val Gln Asn Tyr Arg Lys Thr Pro Thr Val Val 50 55 60 Phe Trp Gln Trp Val Asn Gln Ser Phe Asn Ala Ile Val 65 70 75 <210> SEQ ID NO 5 <211> LENGTH: 1567 <212> TYPE: DNA <213> ORGANISM: human <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (47)..(1015) <400> SEQUENCE: 5 gggcatttgt cccgggacca ggtccacagt tttatgtgtg agcaag atg gag gct 55 Met Glu Ala 1 gac ctg tct ggc ttt aac atc gat gcc ccc cgt tgg gac cag cgc acc 103 Asp Leu Ser Gly Phe Asn Ile Asp Ala Pro Arg Trp Asp Gln Arg Thr 5 10 15 ttc ctg ggg aga gtg aag cac ttc cta aac atc acg gac ccc cgc act 151 Phe Leu Gly Arg Val Lys His Phe Leu Asn Ile Thr Asp Pro Arg Thr 20 25 30 35 gtc ttt gta tct gag cgg gag ctg gac tgg gcc aag gtg atg gtg gag 199 Val Phe Val Ser Glu Arg Glu Leu Asp Trp Ala Lys Val Met Val Glu 40 45 50 aag agc agg atg ggg gtt gtg ccc cca ggc acc caa gtg gag cag ctg 247 Lys Ser Arg Met Gly Val Val Pro Pro Gly Thr Gln Val Glu Gln Leu 55 60 65 ctg tat gcc aag aag ctg tat gac tcg gcc ttc cac ccc gac act ggg 295 Leu Tyr Ala Lys Lys Leu Tyr Asp Ser Ala Phe His Pro Asp Thr Gly 70 75 80 gag aag atg aat gtc atc ggg cgc atg tct ttc cag ctt cct ggc ggc 343 Glu Lys Met Asn Val Ile Gly Arg Met Ser Phe Gln Leu Pro Gly Gly 85 90 95 atg atc atc acg ggc ttc atg ctc cag ttc tac agg acg atg ccg gcg 391 Met Ile Ile Thr Gly Phe Met Leu Gln Phe Tyr Arg Thr Met Pro Ala 100 105 110 115 gtg atc ttc tgg cag tgg gtg aac cag tcc ttc aat gcc tta gtc aac 439 Val Ile Phe Trp Gln Trp Val Asn Gln Ser Phe Asn Ala Leu Val Asn 120 125 130 tac acc aac agg aat gcg gct tcc ccc aca tca gtc agg cag atg gcc 487 Tyr Thr Asn Arg Asn Ala Ala Ser Pro Thr Ser Val Arg Gln Met Ala 135 140 145 ctt tcc tac ttc aca gcc aca acc act gct gtg gcc acg gct gtg ggc 535 Leu Ser Tyr Phe Thr Ala Thr Thr Thr Ala Val Ala Thr Ala Val Gly 150 155 160 atg aac atg ttg aca aag aaa gcg ccg ccc ttg gtg ggc cgc tgg gtg 583 Met Asn Met Leu Thr Lys Lys Ala Pro Pro Leu Val Gly Arg Trp Val 165 170 175 ccc ttt gcc gct gtg gct gcg gct aac tgt gtc aat atc ccc atg atg 631 Pro Phe Ala Ala Val Ala Ala Ala Asn Cys Val Asn Ile Pro Met Met 180 185 190 195 cga cag agg gag ctc ata aag gga atc tgc gtg aag gac agg aat gaa 679 Arg Gln Arg Glu Leu Ile Lys Gly Ile Cys Val Lys Asp Arg Asn Glu 200 205 210 aat gag att ggt cat tcc cgg aga gct gcg gcc ata ggc atc acc caa 727 Asn Glu Ile Gly His Ser Arg Arg Ala Ala Ala Ile Gly Ile Thr Gln 215 220 225 gta gtt att tct cgg atc acc atg tca gct cct ggg atg atc ttg ctg 775 Val Val Ile Ser Arg Ile Thr Met Ser Ala Pro Gly Met Ile Leu Leu 230 235 240 cca gtc atc atg gaa agg ctt gag aaa ttg cac ttc atg cag aaa gtc 823 Pro Val Ile Met Glu Arg Leu Glu Lys Leu His Phe Met Gln Lys Val 245 250 255 aag gtc ctg cac gcc cca ttg cag gtc atg ctg agc ggg tgc ttc ctc 871 Lys Val Leu His Ala Pro Leu Gln Val Met Leu Ser Gly Cys Phe Leu 260 265 270 275 atc ttc atg gtg cca gtg gcg tgt ggg ctt ttc cca cag aaa tgt gaa 919 Ile Phe Met Val Pro Val Ala Cys Gly Leu Phe Pro Gln Lys Cys Glu 280 285 290 ttg cca gtt tcc tat ctg gaa ccg aag ctc caa gac act atc aag gcc 967 Leu Pro Val Ser Tyr Leu Glu Pro Lys Leu Gln Asp Thr Ile Lys Ala 295 300 305 aag tat gga gaa ctt gag cct tat gtc tac ttc aat aag ggt ctc taa 1015 Lys Tyr Gly Glu Leu Glu Pro Tyr Val Tyr Phe Asn Lys Gly Leu 310 315 320 atgccccact tcagcaagga ccagtctatt cccatattca ccagctcctc cttagctacg 1075 tgcacacttg tgtcctcctt cccctttgcc aacaaggcct gaaggccagg gtagattggg 1135 gggtgggaca atgaatgcct catacttaca ccctggtact ggttgattgg acctcagggg 1195 aaaaaagtga aaaagggtag caaaggccaa tgtcttctag ctgcttcctc aacccctgtc 1255 ccctgagaga ccagaagctg aggccctctc agggaggaga catccaagca aatcatttgg 1315 aaaagttagg aaacctttag gattctggtt ccagccaggg ttgaggaaaa gaccttggat 1375 caaaaggaag cttctatacc tctttcttct tcgcttcctc ctctcccaag caatggaaac 1435 ttttacccat gtaattctag ctgaactcag gaaaaagaag ggggaaagga ctctgtcccc 1495 ttggggctca tcacccttcc acatcctcct cctcgtagcc ccctggtcag gcagcttctt 1555 tttttttttt tc 1567 <210> SEQ ID NO 6 <211> LENGTH: 322 <212> TYPE: PRT <213> ORGANISM: human <400> SEQUENCE: 6 Met Glu Ala Asp Leu Ser Gly Phe Asn Ile Asp Ala Pro Arg Trp Asp 1 5 10 15 Gln Arg Thr Phe Leu Gly Arg Val Lys His Phe Leu Asn Ile Thr Asp 20 25 30 Pro Arg Thr Val Phe Val Ser Glu Arg Glu Leu Asp Trp Ala Lys Val 35 40 45 Met Val Glu Lys Ser Arg Met Gly Val Val Pro Pro Gly Thr Gln Val 50 55 60 Glu Gln Leu Leu Tyr Ala Lys Lys Leu Tyr Asp Ser Ala Phe His Pro 65 70 75 80 Asp Thr Gly Glu Lys Met Asn Val Ile Gly Arg Met Ser Phe Gln Leu 85 90 95 Pro Gly Gly Met Ile Ile Thr Gly Phe Met Leu Gln Phe Tyr Arg Thr 100 105 110 Met Pro Ala Val Ile Phe Trp Gln Trp Val Asn Gln Ser Phe Asn Ala 115 120 125 Leu Val Asn Tyr Thr Asn Arg Asn Ala Ala Ser Pro Thr Ser Val Arg 130 135 140 Gln Met Ala Leu Ser Tyr Phe Thr Ala Thr Thr Thr Ala Val Ala Thr 145 150 155 160 Ala Val Gly Met Asn Met Leu Thr Lys Lys Ala Pro Pro Leu Val Gly 165 170 175 Arg Trp Val Pro Phe Ala Ala Val Ala Ala Ala Asn Cys Val Asn Ile 180 185 190 Pro Met Met Arg Gln Arg Glu Leu Ile Lys Gly Ile Cys Val Lys Asp 195 200 205 Arg Asn Glu Asn Glu Ile Gly His Ser Arg Arg Ala Ala Ala Ile Gly 210 215 220 Ile Thr Gln Val Val Ile Ser Arg Ile Thr Met Ser Ala Pro Gly Met 225 230 235 240 Ile Leu Leu Pro Val Ile Met Glu Arg Leu Glu Lys Leu His Phe Met 245 250 255 Gln Lys Val Lys Val Leu His Ala Pro Leu Gln Val Met Leu Ser Gly 260 265 270 Cys Phe Leu Ile Phe Met Val Pro Val Ala Cys Gly Leu Phe Pro Gln 275 280 285 Lys Cys Glu Leu Pro Val Ser Tyr Leu Glu Pro Lys Leu Gln Asp Thr 290 295 300 Ile Lys Ala Lys Tyr Gly Glu Leu Glu Pro Tyr Val Tyr Phe Asn Lys 305 310 315 320 Gly Leu <210> SEQ ID NO 7 <211> LENGTH: 2269 <212> TYPE: DNA <213> ORGANISM: human <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (125)..(1093) <221> NAME/KEY: misc_feature <222> LOCATION: (25)..(25) <223> OTHER INFORMATION: n=A,T,G or C <400> SEQUENCE: 7 gacgcgctcc ggggacgcgc gaggncgccg tggcgggaga agcgtttccg gtggcggcgg 60 aggctgcact gagcgggacc tggcgagcag cgcgggcggc agcccggggg aagcgtccgg 120 gacc atg tct gga gaa cta cca cca aac att aac atc aag gaa cct cga 169 Met Ser Gly Glu Leu Pro Pro Asn Ile Asn Ile Lys Glu Pro Arg 1 5 10 15 tgg gat caa agc act ttc att gga cga gcc aat cat ttc ttc act gta 217 Trp Asp Gln Ser Thr Phe Ile Gly Arg Ala Asn His Phe Phe Thr Val 20 25 30 act gac ccc agg aac att ctg tta acc aac gaa caa ctc gag agt gcg 265 Thr Asp Pro Arg Asn Ile Leu Leu Thr Asn Glu Gln Leu Glu Ser Ala 35 40 45 aga aaa ata gta cat gat tac agg cag gga att gtt cct cct ggt ctt 313 Arg Lys Ile Val His Asp Tyr Arg Gln Gly Ile Val Pro Pro Gly Leu 50 55 60 aca gaa aat gaa ttg tgg aga gca aag tac atc tat gat tca gct ttt 361 Thr Glu Asn Glu Leu Trp Arg Ala Lys Tyr Ile Tyr Asp Ser Ala Phe 65 70 75 cat cct gac act ggt gag aag atg att ttg ata gga aga atg tca gcc 409 His Pro Asp Thr Gly Glu Lys Met Ile Leu Ile Gly Arg Met Ser Ala 80 85 90 95 cag gtt ccc atg aac atg acc atc aca ggt tgt atg atg acg ttt tac 457 Gln Val Pro Met Asn Met Thr Ile Thr Gly Cys Met Met Thr Phe Tyr 100 105 110 agg act acg ccg gct gtg ctg ttc tgg cag tgg att aac cag tcc ttc 505 Arg Thr Thr Pro Ala Val Leu Phe Trp Gln Trp Ile Asn Gln Ser Phe 115 120 125 aat gcc gtc gtc aat tac acc aac aga agt gga gac gca ccc ctc act 553 Asn Ala Val Val Asn Tyr Thr Asn Arg Ser Gly Asp Ala Pro Leu Thr 130 135 140 gtc aat gag ttg gga aca gct tac gtt tct gca aca act ggt gcc gta 601 Val Asn Glu Leu Gly Thr Ala Tyr Val Ser Ala Thr Thr Gly Ala Val 145 150 155 gca aca gct cta gga ctc aat gca ttg acc aag cat gtc tca cca ctg 649 Ala Thr Ala Leu Gly Leu Asn Ala Leu Thr Lys His Val Ser Pro Leu 160 165 170 175 ata gga cgt ttt gtt ccc ttt gct gcc gta gct gct gct aat tgc att 697 Ile Gly Arg Phe Val Pro Phe Ala Ala Val Ala Ala Ala Asn Cys Ile 180 185 190 aat att cca tta atg agg caa agg gaa ctc aaa gtt ggc att ccc gtc 745 Asn Ile Pro Leu Met Arg Gln Arg Glu Leu Lys Val Gly Ile Pro Val 195 200 205 acg gat gag aat ggg aac cgc ttg ggg gag tcg gcg aac gct gcg aaa 793 Thr Asp Glu Asn Gly Asn Arg Leu Gly Glu Ser Ala Asn Ala Ala Lys 210 215 220 caa gcc atc acg caa gtt gtc gtg tcc agg att ctc atg gca gcc cct 841 Gln Ala Ile Thr Gln Val Val Val Ser Arg Ile Leu Met Ala Ala Pro 225 230 235 ggc atg gcc atc cct cca ttc att atg aac act ttg gaa aag aaa gcc 889 Gly Met Ala Ile Pro Pro Phe Ile Met Asn Thr Leu Glu Lys Lys Ala 240 245 250 255 ttt ttg aag agg ttc cca tgg atg agt gca ccc att caa gtt ggg tta 937 Phe Leu Lys Arg Phe Pro Trp Met Ser Ala Pro Ile Gln Val Gly Leu 260 265 270 gtt ggc ttc tgt ttg gtg ttt gct aca ccc ctg tgt tgt gcc ctg ttt 985 Val Gly Phe Cys Leu Val Phe Ala Thr Pro Leu Cys Cys Ala Leu Phe 275 280 285 cct cag aaa agt tcc atg tct gtg aca agc ttg gag gcc gag ttg caa 1033 Pro Gln Lys Ser Ser Met Ser Val Thr Ser Leu Glu Ala Glu Leu Gln 290 295 300 gct aag atc caa gag agc cat cct gaa ttg cga cgc gtg tac ttc aat 1081 Ala Lys Ile Gln Glu Ser His Pro Glu Leu Arg Arg Val Tyr Phe Asn 305 310 315 aag gga ttg taa agcagagagg aaacctctgc agctcattct gccactgcaa 1133 Lys Gly Leu 320 agctggtgta gccatgctgg tgagaaaaat cctgttcaac ctgggttctc ccagttacgg 1193 aaacctttta aagatccaca ttagcctttt agaataaagc tgctacttta acagagcacc 1253 tggcgtgggc caagtgcctg atactccctt acactgaatc atgttatgat ttatagaaat 1313 acctttcctg tagcttttat agtcattgtt tttcaaagac gatataccag ccctcaccca 1373 ggttttaaaa aagcactggt aggcatagaa taggtgctca gtatatggtc agtaaatgtt 1433 ctattgatta tcaatcagtg aaaaaagaaa tctgtttaaa atactgaatt ttcatctcac 1493 tcccattgca aatcaaggag atctcagcag tgaactggga aaatacaaaa gctctgggct 1553 aatctataaa aacttacctg aaatattaag ggcagtttgc ttctagtttg gggattgcgc 1613 tagcccaatg aaggtgatga agcttttgga tttggagggt aaaagctcct tcacacccct 1673 tccaaaagtc agtcacagac cactgcaaca tgccttccct gctggatcat tatatacatt 1733 cagattgtga gtggattgcc ttggttgact tttaatttat tgttttttgt tcttataaag 1793 atgataatct taccttgcag ttattgactt tatattcaat tatttacatc aaataatgaa 1853 ataactgaaa tgtacaaatg tcaaattttg gaagtatatt caataccaat gctgtatgag 1913 tgggctgaat ccagttcatt gttttttttt tggtaagaag tgagactaca gttccagcta 1973 cctacatgtc ttttcttgtc atccttatag atctctttgg ctttcagaaa gatacagtga 2033 taatgtgtgt atgaatcagt cacaatgaat tttacttgaa tattgtatgt tgcattccac 2093 ttcatttgaa aataatgaaa ccatgtacca ctgtttacat catctgtagt gatttcatag 2153 ataatatatt taatatgaca gattatgttt caactctgta gatgtttaac gtcatagaca 2213 gtcggccctc tgtatccgtg agctctatat ctgtgaattc aaccaagttt ggatgg 2269 <210> SEQ ID NO 8 <211> LENGTH: 322 <212> TYPE: PRT <213> ORGANISM: human <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (25)..(25) <223> OTHER INFORMATION: Xaa=A,T,G or C <400> SEQUENCE: 8 Met Ser Gly Glu Leu Pro Pro Asn Ile Asn Ile Lys Glu Pro Arg Trp 1 5 10 15 Asp Gln Ser Thr Phe Ile Gly Arg Ala Asn His Phe Phe Thr Val Thr 20 25 30 Asp Pro Arg Asn Ile Leu Leu Thr Asn Glu Gln Leu Glu Ser Ala Arg 35 40 45 Lys Ile Val His Asp Tyr Arg Gln Gly Ile Val Pro Pro Gly Leu Thr 50 55 60 Glu Asn Glu Leu Trp Arg Ala Lys Tyr Ile Tyr Asp Ser Ala Phe His 65 70 75 80 Pro Asp Thr Gly Glu Lys Met Ile Leu Ile Gly Arg Met Ser Ala Gln 85 90 95 Val Pro Met Asn Met Thr Ile Thr Gly Cys Met Met Thr Phe Tyr Arg 100 105 110 Thr Thr Pro Ala Val Leu Phe Trp Gln Trp Ile Asn Gln Ser Phe Asn 115 120 125 Ala Val Val Asn Tyr Thr Asn Arg Ser Gly Asp Ala Pro Leu Thr Val 130 135 140 Asn Glu Leu Gly Thr Ala Tyr Val Ser Ala Thr Thr Gly Ala Val Ala 145 150 155 160 Thr Ala Leu Gly Leu Asn Ala Leu Thr Lys His Val Ser Pro Leu Ile 165 170 175 Gly Arg Phe Val Pro Phe Ala Ala Val Ala Ala Ala Asn Cys Ile Asn 180 185 190 Ile Pro Leu Met Arg Gln Arg Glu Leu Lys Val Gly Ile Pro Val Thr 195 200 205 Asp Glu Asn Gly Asn Arg Leu Gly Glu Ser Ala Asn Ala Ala Lys Gln 210 215 220 Ala Ile Thr Gln Val Val Val Ser Arg Ile Leu Met Ala Ala Pro Gly 225 230 235 240 Met Ala Ile Pro Pro Phe Ile Met Asn Thr Leu Glu Lys Lys Ala Phe 245 250 255 Leu Lys Arg Phe Pro Trp Met Ser Ala Pro Ile Gln Val Gly Leu Val 260 265 270 Gly Phe Cys Leu Val Phe Ala Thr Pro Leu Cys Cys Ala Leu Phe Pro 275 280 285 Gln Lys Ser Ser Met Ser Val Thr Ser Leu Glu Ala Glu Leu Gln Ala 290 295 300 Lys Ile Gln Glu Ser His Pro Glu Leu Arg Arg Val Tyr Phe Asn Lys 305 310 315 320 Gly Leu 

1. An isolated nucleic acid molecule selected from: (a) nucleic acid molecules comprising a nucleotide sequence as shown in SEQ ID NO: 1, 3, 5 or 7; (b) nucleic acid molecules comprising a nucleotide sequence capable of hybridizing, under stringent hybridization conditions, to a nucleotide sequence complementary to the polypeptide coding region of a nucleic acid molecule as defined in (a); and (c) nucleic acid molecules comprising a nucleic acid sequence which is degenerate as a result of the genetic code to a nucleotide sequence as defined in (a) or (b).
 2. An isolated polypeptide encoded by the nucleic acid molecule according to claim
 1. 3. The isolated polypeptide according to claim 2 having an amino acid sequence shown as SEQ ID NO: 2, 4, 6, or 8 in the Sequence Listing.
 4. A vector harboring the nucleic acid molecule according to claim
 1. 5. A replicable expression vector which carries and is capable of mediating the expression of a nucleotide sequence according to claim
 1. 6. A cultured host cell harboring a vector according to claim 4 or
 5. 7. A process for production of a polypeptide, comprising culturing a host cell according to claim 6 under conditions whereby said polypeptide is produced, and recovering said polypeptide.
 8. A method for identifying an agent capable of modulating a nucleic acid molecule according to claim 1, comprising (i) providing a cell comprising the said nucleic acid molecule; (ii) contacting said cell with a candidate agent; and (iii) monitoring said cell for an effect that is not present in the absence of said candidate agent. 